DESCRIPTION: This application is for renewal of a grant that focuses broadly on understanding how the sterol biosynthetic pathways is regulated in Saccharomyces. The regulation of cholesterol levels is a key component of cardiovascular disease and past studies from this grant have revealed several novel dimensions, which were later extended to human cells. Moreover, the past work has uncovered a fundamental biochemical connection between sterol biosynthesis and the function of the Ras oncoprotein, which is second only to p53 for the frequency of being involved in human cancer. Ras is covalently coupled to a prenyl lipid intermediate of sterol biosynthesis, and these discoveries spurred development of potential anti cancer therapies that inhibit prenyl-transferases. The experiments proposed here continue along these lines with three foci. First, experiments will define regulatory sites and proteins through which sterol levels control transcription of genes in this pathway. Using a complete set of fluorescent-based reporters for each gene in the pathway, the entire pathway will be evaluated for its response to sterol levels, with there being some hints already that more than one regulon may be at work. A comprehensive test of the role of each of 7 oxysterol binding protein homologs in yeast, whose mammalian homolog is proposed to mediate sterol regulation in mammals, will be performed. The second focus is on protein prenylation and Ras. A novel model for how Ras protein is trafficked to the plasma membrane, which emerged from the last grant period, will be tested to determine whether Ras is transported from the ER, the site of several Ras processing enzymes, to the plasma membrane on the outside of secretory vesicles. In addition, a complete description of the substrate specificity determinants of CAAX proteases, which were also discovered in the last grant period, will also be obtained for both the protein and lipid substrates. In the process, potentially every prenylated protein in the cell will be identified, and for each protein, the CAAX protease responsible for processing it will be identified. The third focus will be whether CAAX protease of human Ras is important for its oncogenic function. We will develop antibodies that inhibit the CAAX protease of a vertebrate and determine in vivo whether inhibition of this enzyme blocks the maturation and activation of Ras. In addition, proteins that bind to the C-terminus of Ras will be tested to learn whether these proteins mediate the membrane association of Ras.